Molybdenum nitrogenase of Azotobacter chroococcum. Tight binding of MgADP to the MoFe protein.
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ABSTRACT:
SUBMITTER: Miller RW
PROVIDER: S-EPMC1133492 | biostudies-other | 1989 Nov
REPOSITORIES: biostudies-other
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Project description:The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08 Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the β-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.
| S-EPMC4321486 | biostudies-literature
Project description:A molybdenum L-edge X-ray absorption spectroscopy (XAS) study is presented for native and oxidized MoFe protein of nitrogenase as well as Mo-Fe model compounds. Recently collected data on MoFe protein (in oxidized and reduced forms) is compared to previously published Mo XAS data on the isolated FeMo cofactor in NMF solution and put in context of the recent Mo K-edge XAS study, which showed a MoIII assignment for the molybdenum atom in FeMoco. The L3-edge data are interpreted within a simple ligand-field model, from which a time-dependent density functional theory (TDDFT) approach is proposed as a way to provide further insights into the analysis of the molybdenum L3-edges. The calculated results reproduce well the relative spectral trends that are observed experimentally. Ultimately, these results give further support for the MoIII assignment in protein-bound FeMoco, as well as isolated FeMoco.
| S-EPMC4510703 | biostudies-literature
Project description:A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.
| S-EPMC1135427 | biostudies-other
Project description:Biosynthesis of nitrogenase P-cluster has attracted considerable attention because it is biologically important and chemically unprecedented. Previous studies suggest that P-cluster is formed from a precursor consisting of paired [4Fe-4S]-like clusters and that P-cluster is assembled stepwise on MoFe protein, i.e., one cluster is assembled before the other. Here, we specifically tackle the assembly of the second P-cluster by combined biochemical and spectroscopic approaches. By using a P-cluster maturation assay that is based on purified components, we show that the maturation of the second P-cluster requires the concerted action of NifZ, Fe protein, and MgATP and that the action of NifZ is required before that of Fe protein/MgATP, suggesting that NifZ may act as a chaperone that facilitates the subsequent action of Fe protein/MgATP. Furthermore, we provide spectroscopic evidence that the [4Fe-4S] cluster-like fragments can be converted to P-clusters, thereby firmly establishing the physiological relevance of the previously identified P-cluster precursor.
| S-EPMC1965529 | biostudies-literature
Project description:Nitrogenase catalyzes the six electron/six proton reduction of N2 to two ammonia molecules at a complex organometallocluster called "FeMo cofactor." This cofactor is buried within the alpha-subunit of the MoFe protein, with no obvious access for substrates. Examination of high-resolution X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent-exposed surface to a specific face of FeMo cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate reduction kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters were established for the reduction of the substrates N2, acetylene, azide, and propyne. For each MoFe protein, V (max) values for the different substrates were found to be nearly unchanged when compared with the values for the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, the K(m) values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared with the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water-filled channel functioning as a substrate channel in the MoFe protein.
| S-EPMC2819195 | biostudies-literature
Project description:Another iron in the fire: X-ray anomalous diffraction studies on the nitrogenase MoFe protein show the presence of a mononuclear iron site, designated as Fe16, which was previously identified as either Ca(2+) or Mg(2+). The position of the absorption edge indicates that this site is in the oxidation state +2. The high sequence conservation of the residues coordinated to Fe16 emphasizes the potential importance of the site in nitrogenase.
| S-EPMC3891402 | biostudies-literature
Project description:The engineering of transgenic organisms with the ability to fix nitrogen is an attractive possibility. However, oxygen sensitivity of nitrogenase, mainly conferred by the reductase component (NifH)2 , is an imminent problem. Nitrogenase-like enzymes involved in coenzyme F430 and chlorophyll biosynthesis utilize the highly homologous reductases (CfbC)2 and (ChlL)2 , respectively. Chimeric protein-protein interactions of these reductases with the catalytic component of nitrogenase (MoFe protein) did not support nitrogenase activity. Nucleotide-dependent association and dissociation of these complexes was investigated, but (CfbC)2 and wild-type (ChlL)2 showed no modulation of the binding affinity. By contrast, the interaction between the (ChlL)2 mutant Y127S and the MoFe protein was markedly increased in the presence of ATP (or ATP analogues) and reduced in the ADP state. Upon formation of the octameric (ChlL)2 MoFe(ChlL)2 complex, the ATPase activity of this variant is triggered, as seen in the homologous nitrogenase system. Thus, the described reductase(s) might be an attractive tool for further elucidation of the diverse functions of (NifH)2 and the rational design of a more robust reductase.
| S-EPMC7317204 | biostudies-literature
Project description:1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes for conventional nitrogenase (nifHDK) was separated into two components: an Fe-containing protein and a vanadoprotein. 2. The larger protein was purified to homogeneity by the criterion of electrophoresis of 10% (w/v) acrylamide gels in the presence of SDS. Two types of subunit, of Mr 50,000 and 55,000, were present in equal amounts. 3. The protein had an Mr of 210,000 and contained 2 V atoms, 23 Fe atoms and 20 acid-labile sulphide groups per molecule. The Mo content was less than 0.06 g-atom/mol. All the common amino acids were present, with a predominance of acidic residues. Ultracentrifugal analysis gave a maximum sedimentation coefficient of 9.7 S and a symmetrical boundary at 5 mg of protein X ml-1; dissociation occurred at lower concentrations. The specific activities (nmol of product/min per mg of protein), when assayed under optimum conditions with the complementary Fe protein from this strain, were 1348 for H2 evolution, 350 for NH3 formation and 608 for acetylene reduction. Activity was O2-labile, with a t1/2 of 40 s in air. At low temperatures the dithionite-reduced protein showed e.p.r. signals at g = 5.6, 4.35, 3.77 and 1.93, consistent with an S = 3/2 ground state with an additional S = 1/2 centre giving rise to the feature at g = 1.93. The u.v. spectra of dithionite-reduced and thionine-oxidized protein were very similar. Oxidation resulted in a general increase in absorbance in the visible region. The shoulder at 380 nm in the spectrum of reduced protein was replaced with shoulders near 330 nm and 420 nm on oxidation.
| S-EPMC1147972 | biostudies-other
Project description:1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes of Monitrogenase (nifHDK) was associated with a V + Fe-containing protein and an Fe-containing protein [Robson, Eady, Richardson, Miller, Hawkins & Postgate (1986) Nature (London) 322, 388-390; Eady, Robson, Richardson, Miller & Hawkins (1987) Biochem. J. 244, 197-207]. 2. The Fe protein was purified to homogeneity by the criterion of Coomassie Blue staining after electrophoresis in 10% or 17% (w/v) polyacrylamide gels in the presence of SDS. One type of subunit, of Mr 32,000 +/- 2000, was found. 3. The native protein had an Mr of 62,500 +/- 2500 and contained approximately 4 Fe atoms and 4 acid-labile sulphide groups per molecule. The amino acid composition was similar to those of other purified Fe proteins, and, characteristically, tryptophan was absent. The specific activities (nmol of protein/min per mg of protein) when assayed under optimum conditions with the VFe protein from this strain were 1211 for H2 evolution under Ar, 337 for NH3 from N2 formation and 349 for C2H2 reduction. Activity of the Fe protein was O2-labile with a t1/2 of 36 s in air. At low temperatures the dithionite-reduced protein exhibited e.p.r. signals consistent with the presence of both S = 1/2 and S = 3/2 spin states. These signals were similar to those given by other nitrogenase Fe proteins, as were the changes in their line shape that occurred in the presence of MgATP or MgADP. The absorbance spectra showed that an increase in absorption occurred in the visible range on reversible oxidation of the dithionite-reduced protein. The oxidized-minus-reduced epsilon 420 was 6000 M-1.cm-1.
| S-EPMC1135386 | biostudies-other
Project description:The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.
| S-EPMC1135657 | biostudies-other